Herpesviruses have been found in most animal species and approximately 100 herpesviruses have been at least partially characterized. These include species causing diseases in humans, horses, cattle, pigs, and chickens. In humans, the seven herpesviruses that have been thus far isolated are viewed as important causes of human morbidity and mortality. [Whitley, R. J., Virology (1990) New York, Raven Press]. Thus, the availability of methods for detecting infectious herpesviruses has become increasingly important.
The eight known human herpesviruses are herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV6), human herpes virus 7 (HHV7), and herpes human virus 8. The herpesviruses differ with respect to the diseases they cause. HSV-1 infection produces skin vesicles or mucosal ulcers generally limited to the oropharynx; HSV-2 produces mucocutaneous lesions generally in the genital region; HCMV can infect monocytes and a number of organ systems including salivary glands, kidneys, liver and lung; VZV causes the diseases of chickenpox and shingles; EBV infects the oropharyngeal epithelium and B lymphocytes; and HHV6, HHV7 and HHV8 infect mononuclear cells and, in children, produce skin eruptions (Roseola Infantum). (Virology, Fields and Knipe, Eds., Raven Press, pp. 1795-2062).
The human herpesviruses HSV-2, HHV6 and HHV7 have been partially sequenced and HSV-1, HCMV, EBV and VZV have been completely sequenced. (McGeoch et al., Nucleic Acids Res. 14: 1727-1745, 1986 and McGeoch et al., J. Gen. Virol 69: 1531-1574, 1988; Chee et al., DNA Seq. 2: 1-12, 1991; Baer et al., Nature 310: 207-211, 1984; Davidson and Scott, J Gen Virol 67: 1759-1816, 1986). Furthermore, the DNAs of the human herpesviruses have been found to be similar, but not identical. For example, all known human herpesviruses have DNA sequences that encode a ribonucleotide reductase enzyme, which contains a large subunit referred to as Infected-Cell Protein (ICP). In HSV-1 this DNA sequence encodes a ribonucleotide reductase large subunit (ICP6) that has 38% homology at the N-terminal portion and 93% homology at the C-terminal portion of the corresponding HSV-2 protein (ICP10). (Nikas et al, Proteins: Structure, Function, and Genetics 1: 376-384, 1986). The corresponding ribonucleotide reductase large subunit in VZV shows between 43% and 53% homology beginning at HSV-1 amino acid 384 and VZV amino acid 16. Id.
Herpes simplex viruses Types 1 and 2 (hereinafter referred to collectively as HSV) infect a large number of individuals each year. Primary infection of immunocompetent patients with HSV usually leads to a mucocutaneous syndrome such as herpes labialis (HSV-1) or herpes genitalis (HSV-2), the latter being one of the most common sexually transmitted diseases today. Infection with HSV can also cause more serious infections, the most serious of which are sight-threatening keratitis and life-threatening encephalitis. Moreover, HSV related disease in immunocompromised individuals such as newborns, leukemia patients, organ transplant recipients and AIDS patients has become an increasingly prevalent and difficult problem.
Significant advances have been made in the treatment of HSV infections in the past decade. These advances in antiviral therapy have expanded the role of the diagnostic virology laboratory and have identified the need for more sensitive, accurate and rapid diagnostic tests to assist in the early diagnosis of HSV infections.
Various tests are presently available for the diagnosis of HSV infections. Most involve the detection of viral antigens or intact infectious virus. Antigen detection assays offer the advantage of rapidity and specificity, but lack the necessary sensitivity. [Kowalski, R. P. and Gordon, Y. J., Opthal. (1989) 96: 1583-1586]. The most reliable test to detect infectious herpesvirus involves inoculation of specimens onto tissue culture cells followed by detection of infectious virus by microscopically observing a characteristic cytopathic effect.
Although HSV is a relatively easy virus to culture as it replicates on a wide variety of continuous cell lines, virus propagation in tissue culture can be slow and expensive. Recently, improved techniques have been developed for the detection of viruses from clinical specimens. The shell vial technique, for instance, has greatly increased the sensitivity and the rapidity of HSV detection. When this method is combined with antigen detection by immunohistochemistry, HSV can be positively identified within 24 hours in the majority of cases. Gleaves et al., J. Clin. Mircro. (1985) 21: 29-32; Ziegler et al., J. Clin. Micro. (1985) 26: 2013-2017. While this type of assay is preferred in diagnostic virology applications, it is labor intensive and a significant number of specimens are not identified as positive until after 48 hours.
Another recent technological advance, polymerase chain reaction (PCR) technology, presents a promising tool for the detection of HSV particularly in cerebrospinal fluid specimens, but this technology detects viral nucleic acid and not infectious virus. (Puchhammer-Stockl, et al., J. Med. Virol. (1990) 32: 77-82). The detection of infectious virus is preferred because it definitively indicates that there is an ongoing viral infection with active viral replication. PCR detection of viral nucleic acid may only be indicative of the presence of a remnant of a past infection or the presence of a latent infection.
The commercial availability of HSV type-specific monoclonal antibodies has enhanced the ability of the diagnostic virology practice to provide a result which identifies HSV-1 (majority of oral infections) or HSV-2 (majority of genital infections) from a clinical specimen. However, the 48 hour procedure is time consuming, expensive and labor intensive in that it requires duplicate cultures be inoculated with clinical specimen. After 24-48 hours, one culture is reacted with antibody to HSV-1 antigen and the duplicate culture is reacted with antibody to HSV-2 antigen. After multiple steps and incubation periods, the two cell cultures are evaluated microscopically to determine which of the two antibody reagents produced a positive detection signal on the infected cell monolayer.
Previous scientific studies involving herpesviruses have used susceptible cell lines transfected with a chimeric DNA construct containing a marker gene in transient assays to study various aspects of the virus such as the regulation of gene expression during viral replication. [Flanagan W. M. and Wagner, K. K., Virus Genes 1: 1: 61-71 (1987)]. These studies have not, however, described a DNA construct stably integrated into the chromosome of a stable cultured cell line which is suitable for the diagnostic detection and quantification of a herpesvirus in a specimen with the requisite sensitivity and specificity for a clinical diagnostic assay.
A method for detecting infectious HIV in a specimen has been disclosed that utilizes a genetically engineered cell line containing a chimeric gene having the E. coli lacZ gene associated with the HIV-1 LTR promoter. [Rocancourt, et al., J. Virol. (1990) 64: 2660-2668; Kimpton, J. and Emerman, M., J. Virol. (1992) 66: 4: 2232-2239]. Although these cell lines may be useful for detecting HIV in a specimen, they are not suitable for diagnostic virology assays because of their lack of specificity. It is well-known that the HIV-1 LTR promoter used in the DNA construct of these studies to cause expression of the reporter gene is not specific for HIV and that other viruses cause expression of the reporter gene if present in the specimen. In particular, the presence of HSV or cytomegalovirus in the specimen causes activation of the LTR promoter and subsequent expression of the reporter gene even in the absence of HIV in a specimen. [Mosca, J. D., et al., Nature (1987) 325: 67 70; Mosca, J. D., et al., Proc. Natl. Acad. Sci. (1987) 84: 7408-7412; Popik and Pitha, Proc. Natl. Acad. Sci. (1981) 88: 9572-9577]. If such a cell line were used in a diagnostic assay, it could lead to the erroneous diagnosis of the presence of HIV in a specimen when in fact the specimen contained a different virus. Thus, the lack of specificity in cell lines prepared to detect HIV in a specimen prevents their use in a diagnostic assay which requires specificity.
Recently, a method for detecting infectious HSV in a specimen has been disclosed that utilizies a genetically engineered cell line containing a chimeric gene having the E. coli lacZ gene associated with the HSV-1 promoter region for the viral ribonucleotide reductase, known as ICP6 [U.S. Pat. No. 5,418,132 to Olivio, herein incorporated by reference and Stabell et al., J. Clin. Microbiol. 31: 2796-2798 (1993)]. When either HSV-1 or HSV-2 infects this line, .beta.-galactosidase (the product of the lacZ gene) is made and accumulates in the cytoplasm of induced cells. While this genetically engineered cell line allows for the detection of infectoius HSV in a sample, it cannot distinguish between the HSV-1 and HSV-2 types.
A need exists, therefore, for methods of detecting infectious virus, including herpesvirus, in a specimen that provides rapid detection in a cost efficient manner, while also providing the sensitivity and specificity necessary for a diagnostic assay. Ideally, the diagnostic assay would allow the identification of different types of virus (e.g., HSV-1 and HSV-2).